Antimicrobial resistance of Staphylococcus spp. in swine farming: a challenge for one health Resistência antimicrobiana de Staphylococcus spp. na suinocultura: desafio para a saúde única

Article history The inadequate and excessive use of antimicrobial agents in pig farming has contributed to the emergence and increase of resistance to antibiotics in both bacteria related to infectious processes in these animals as those that constitute their own microbiota. This conduct also causes the dissemination of these microorganisms throughout the pig production chain, causing damages to health of consumers of their meat and processed-meat products. The effect of excess use of these medicines can even reach and compromise other ecosystems. Methicillin-resistant Staphylococcus (MRS) stands out among bacterium species of interest to the public health. They emerged as important zoonotic pathogens, whose evolution generated different virulence and mechanisms of resistance to antimicrobial agents and has been associated to high use of these medicines in pig farming. The development of resistance to antibiotics in Staphylococcus spp., especially the expression of the gene mecA, and their interrelation with pig farming are aspects considered in this work. The emergence and global presence of MRS in pig farming denote the important epidemiological involvement of these animal species in the dissemination of these microorganisms, and the occurrence of infections in humans and animals in the whole world. This is a scenario that requires attention by public health agencies and should not be overlooked. Received 16 June 2019 Accepted 13 October 2019


INTRODUCTION
Changes in animal-origin food production have required high use of antimicrobial agents to maintain the health of animals or increase their yield. However, this excessive use has favored the increase of global Antimicrobial Resistance (AMR), which is a great threat to human and animal health, denoting dangers to modern human and veterinary medicines, and hinders the food and environmental safety (MOREL, 2019;REGITANO;LEAL, 2010).
Pig farming stands out among the food production chains that use antimicrobial agents inadequately or abusively (AGUILAR et al., 2015;BARCELLOS et al., 2009;SANTOS et al., 2009).
The excess use of these medicines can be exemplified by the case of Staphylococcus spp., whose evolution generated different mechanisms of resistance to antibiotics has enabling the emergence of multiresistant strains. This fact has been related to their colonization in pigs and the increasing use of antibiotics in these animals (BUTAYE; ARGUDÍN; SMITH, 2016).
The present work discusses the use of antimicrobial agents in pig farming and the emergence of the mec determinant as the main resistance mechanism of Staphylococcus spp. and its transmissibility between these animals and humans.

PIG FARMING AND USE OF ANTIBIOTICS
Pig farming is changing from extensive systems to more intensive managements of production because of the need for increasing yield (SEAB, 2013;USDA, 2015). As a consequence, there is a higher contact between animals in this production system, which contributes to an increase in occurrence and dissemination of infectious diseases and also increases the stress level of the animals, affecting negatively the defense mechanisms of their immune system (BARCELLOS et al., 2009).
Several strategies have been used to minimize the risks of the intensive production systems, including: medication programs and rigorous norms for cleaning, disinfection, and sanitary void between the lots, and the use of antimicrobial agents (AGUILAR et al., 2015;BARCELLOS et al., 2009;SANTOS et al., 2009).
Most pig farms use antimicrobial agents throughout the development of the animal until the slaughter, which is not limited only to the infection therapeutics in these animals. The prophylactic use to control diseases and promote the growth of animals increases the use of these medicines, which are also been adopted to the detriment of basic hygiene norms and conducts (COMPASSION IN WORLD FARMING, 2011;FDA, 2018;FERREIRA, 2014).
Global data estimated that the consumption of antimicrobial agents for animal production in 2013 was above 131,000 Mg, with an increase of 50% for 2030 (VAN BOECKEL et al., 2017). These data show the high use of these medicines, which became essential for intensive production systems. This use can reach 60% to 80 % of the total antimicrobial agents used in the United Kingdom (UK), some countries of the European Union (EU), and in the United States (USA) (AGUILAR et al., 2015;BARCELLOS et al., 2009;SANTOS et al., 2009).
The high use of these medicines in pig farming generates a high selection pressure, favoring the emergence and increase of resistance to antibiotics in microorganisms involved with infectious diseases in these animals and also in their own microbiota (BARTLETT; GILBERT;SPELLBERG, 2013;DARWISH et al., 2013;MARSHALL;LEVY, 2011;VAN BOECKEL et al., 2015).
The inadequate use of these medicines also has consequences to the environment, since 90.0% of antimicrobial agents administered to animals are eliminated by urine and feces, and the dispersion of these products can compromise some ecosystems (MOREL, 2019;REGITANO;LEAL, 2010).
Another consequence of the inadequate use of antimicrobial agents in swine farming is their effects on the health of humans that consume the meat or processed-meat products from this animal species. These effects can be due to: a) possible presence of antibiotic residues that can trigger allergic reactions or other diseases, which can even cause death; and b) presence of resistant bacteria in the food (DARWISH et al., 2013;RAKOTOHARINOME et al., 2014;REGITANO;LEAL, 2010;SERI, 2013).
Restrictions in the use of these medicines started to be considered in England, a pioneer country that developed regulations in the 1960's to avoid the consequences of inadequate or excessive application of antimicrobial agents in livestock. According to the Swann Report, the English government recommended that antibiotics used for both human and veterinary therapeutics should no longer be used as growth promoters (KIRCHHELLE, 2018a).
This directive was followed by other countries until January 2008, when the EU made the prohibition of this additive official by the regulation CE Nº 1831/2003DUCATELLE;IMMERSEEL, 2011;NÉVOA et al., 2013;NOSCHANG et al., 2017). Since then, consumer and producing countries have been advancing and moving back on regulations for the use of antimicrobial agents in animal production (KIRCHHELLE, 2018b).
In addition to some individual initiatives, the increasing reports of resistance to antibiotics in the world made the World Health Organization (WHO) to implement a global task force in 2015, involving the Food and Agriculture Organization (FAO), World Organization for Animal Health (OIE), and United Nations (UN), to gather data on the consumption and the impact of use of antimicrobial agents, and to define and assist the execution of strategies to contain the advance of resistance to antibiotics in the whole world (FAO, 2016;OIE, 2019;WHO, 2015;WHO, 2017).
The use of antimicrobial agents as prophylactics and growth promoter in Brazil is not prohibited. However, the Brazilian Ministry of Agriculture and Livestock and Supply (MAPA), considering the history of international concerns (OMS, FAO, Codex Alimentarius) about the increasing resistance to antimicrobial agents, vetoed the use of several of these substances as additive for performance improvement in animals used for food production (BRASIL, 2018;CARDOSO, 2019). Table 1 presents the main Brazilian regulations related to the prohibition of use of antimicrobial agents in animal production. The main antibiotics used in the therapeutics of food animals, including pig, are: tetracycline, penicillin, fluoroquinolone, streptomycin, erythromycin, nystatin, tyrosine, virginiamycin, and sulfonamides; some of which are also used for treatments of infections in humans. Therefore, the presence of multiresistant bacteria can hinder the efficacy of therapeutics of infectious diseases either in humans or in animals (COMPASSION IN WORLD FARMING, 2011;FDA, 2018) .
There are reports of dissemination of several resistant microorganisms throughout the pig meat production cycle. Among these microorganisms, some stand out by their high morbidity, multiresistance, and involvement in outbreaks, including Staphylococcus aureus, Enterococcus spp., Escherichia coli, and Salmonella spp., which cause infections in humans and in animals (HAMMERUM et al., 2014;OLIVEIRA et al., 2011;SFACIOTTE et al., 2015).

Staphylococcus spp.
The genus Staphylococcus has 54 species and 28 subspecies described as Gram-positive coccus, nonmobile, with diameters of 0.5 the 1.5 μm, and producers of the catalase enzyme, which tend to form clusters similar to grape bunches (PARTE, 2018; SCHLEIFER; BELL, 2015).
In addition to the several virulence factors that S. aureus can express, it shows a high versatility for emergence of different determinants of resistance to antibiotics. This capacity has challenging the empirical treatment and control of Staphylococcus infections (FOSTER, 2017;MARQUES et al., 2008;RATTI;SOUSA, 2009).

Methicillin-resistant Staphylococcus spp.
The emergence of successive mechanisms of resistance in Staphylococcus spp. to different classes of antimicrobial agents have been seen in genetic changes due to mutations or horizontal transference of genes contained in mobile accessory genetic elements, such as plasmids, transposons, and pathogenicity genomic islands (JENSEN; LYON, 2009;MUNITA;ARIAS, 2016;PARTRIDGE et al., 2018).
The antibiotic resistance developed by Staphylococcus spp. to several antimicrobial agents used in therapeutics is expressed by different biochemical mechanisms and involves several genes that can codify a phenotype of resistance to a specific medicine. This diversity has been found in strains of Staphylococcus spp. of animal and human origin. In addition, some genes of resistance can be shared between animals and humans (ARGUDIN et al., 2017;KADLEC et al., 2012;REYGAERT, 2013;WENDLANDT et al., 2013).
The different biochemical resistance mechanisms of these microorganisms to antibiotics and the profile of some related genes are presented in Table 2. Table 2. Main mechanisms of acquired resistance to antimicrobials observed in Staphylococcus spp. strains.

Mechanism Affected Antimicrobial Class (examples of genes involved)
Enzymatic Inactivation Beta-lactams (blaZ), Aminoglycosides (aacA-aphD) and Phenicols The evolution of Staphylococcus spp. for resistance to antibiotics emerged with the use of sulfonamides in the late 1930's and, currently, adapted strains are maintaining resistant to new different antibiotics used in therapeutics for infections caused by these microorganisms (ADALETI et al., 2010;RINCÓN et al., 2014;SANTOS et al., 2007;VELÁZQUEZ-GUADARRAMA et al., 2010).
Resistance to beta-lactam antibiotics is among the mechanisms of antimicrobial resistance that species of this genus can have, and became the most studied (HAMILTON et al., 2017;LAKHUNDI;ZHANG;PEACOCK;PATERSON, 2015).
The beta-lactam antibiotics are bactericidal antimicrobial agents, represented by penicillin, penicillin combined with inhibitors of beta-lactamase enzymes, semisynthetic penicillin, and cephalosporins (PLATA; ROSATO; WEGRZYN, 2009). These compounds bind to proteins present in the bacterial cell membrane known as penicillin-binding proteins (PBPs), which have enzymatic functions involved with the establishment of cross-links between the peptidoglycan molecules, resulting in the synthesis of the bacterium wall. Thus, these medicines prevent these bindings and activate the function of autolysins enzymes, breaking the cell wall and leading to the death of the bacterium (FOSTER, 2017;PLATA;ROSATO;WEGRZYN, 2009;RATTI;SOUSA, 2009).
Resistance to penicillin G by S. aureus strains emerged in 1944 because of the production of penicillinases or betalactamases enzymes, which hydrolyze the beta-lactam ring of this medicine inactivating the penicillin (COHEN, 1986). In the late 1950's, the percentage of resistant S. aureus strains to this antimicrobial that were isolated in the hospital environments reached 80% (LYON; SKURRAY, 1987).
Penicillins resistant to beta-lactamases were produced in the 1960's to solve this problem; this was a new group of beta-lactam semisynthetic antibiotics that include oxacillin and the methicillin (FRENCH, 2010). However, the first resistant S. aureus strains to methicillin emerged after only one year of using these antimicrobials; they were termed methicillin-resistant Staphylococcus aureus (MRSA) (JEVONS, 1961;LAKHUNDI;ZHANG;. The resistance to oxacillin is determined by a mobile genetic element termed Staphylococcal cassette chromosome mec (SCCmec), which has the mecA generesponsible for the resistance to beta-lactam antibiotics (HIRAMATSU et al., 2013).
The insertion of SCCmec in the chromosome of methicillin-sensitive Staphylococcus aureus (MSSA) strains is the key event for the emergence of MRSA. SCCmec is a mobile genetic element that is highly diverse in its structural organization and genetic content; three regions are used for its classification: a) mec complex, which includes the mecA gene and can include the regulatory genes mecI and mecR1, which are involved with the expression of mecA gene; and can include the sequence IS431; b) ccr complex, which can include the ccrAB or ccrC genes, which codify recombinase enzymes that control integration and excision in the genome that will host the SCCmec; thus, they are responsible for the mobility of the mec determinant; c) J regions (J1, J2, and J3), which consist of non-essential elements to SCCmec, but, in some cases, they have carried additional determinants of resistance to antibiotics. SCCmec is integrated to a specific site (attBscc) located near the origin of replication of S. aureus (EL-HAMID, 2016;HIRAMATSU et al., 2013;TURLEJ;HRYNIEWICZ;EMPEL, 2011). Figure 1 shows the basic structure of SCCmec. SCCmec types I, II, and III are frequently described in MRSA strains associated to hospital infections-hospitalassociated methicillin resistant Staphylococcus aureus (HA-MRSA).
They present a mec determinant of relatively large size (34.3 to 66.9 Kb) and carry several markers of resistance to antibiotics. MRSA containing SCCmec types IV and V are characteristic of strains that emerged in the community and are termed community-associated methicillin resistant Staphylococcus aureus (CA-MRSA); these strains have a mec determinant of smaller size (20.9 to 28 Kb) and commonly do not have genes that codify resistance to other non-beta-lactam antimicrobial agents; thus, these MRSA are sensitive to most antimicrobial agents belonging to other classes (DEURENBERG et al., 2007;LAKHUNDI;ZHANG, 2018).
HA-MRSA strains emerged in hospitals in the 1960's, and the CA-MRSA strains started to be described in the 1980's. These MRSA were spread in hospital environments and in communities around the world. The coexistence of these microorganisms in these ecosystems and their overcoming of ecological barriers have been reported, with infections or colonization in pets and production animals (BARBER, 1961;CHAMBERS, 1988;EVANGELISTA;OLIVEIRA, 2015;FIGUEIREDO;FERREIRA, 2014;KAYSER;MAK, 1972;KLEIN et al., 2013).
The first report of MRSA strains causing infections in animals was described in cases of bovine mastitis in Belgium (DEVRIESE; VAN DAMME;FAMEREE, 1972). Since then, sporadic reports and outbreaks affecting pets and production animals, such as horses, birds, pigs, dogs, and cats, have been reported around the world (CUNY et al., 2010;FITZGERALD, 2012;PRICE et al., 2012). The presence of HA-MRSA or CA-MRSA in raw or processed meat foods has also been reported, denoting the dissemination potential of these microorganisms (AIRES-DE-SOUSA, 2017;COSTA et al., 2015).
MRSA strains from animals but capable of colonize humans are termed livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA); they have drawn attention since 2005 due to concerns regarding public health, especially the MRSA strains termed LA-MRSA ST398 (FITZGERALD, 2012;PEETERS et al., 2015;PETON;LE LOIR, 2014). This MRSA clone emerged in pigs and has been described colonizing or causing infections in humans, production animals (bovine, birds, and equines), pets (dogs and cats), and wild species in different countries, denoting its high-dissemination potential (BUTAYE; ARGUDÍN;SMITH, 2016;LAKHUNDI;ZHANG, 2018;LIMA et al., 2017;PEETERS et al., 2015;SMITH, 2015). García-Álvarez et al. (2011) found MRSA strains in bovine milk, in which the presence of the mecA gene was not confirmed by polymerase chain reaction (PCR), which is commonly used for identification of MRSA of different origins; or by agglutination tests for PBP2a. After further analysis, García-Álvarez et al. (2011) found differences in nucleotide sequences of the mecA gene in the strains and characterized a new mecA gene homologous to that widely known, which they termed mecALGA251 and lately was termed mecC. The diversity found in the SCCmec determinant of this microorganisms allowed its classification as SCCmec type XI by Ito et al. (2012), who described the presence of the genetic determinant mecC in S. aureus strains isolated from human specimens.
The identification of MRSA strains that have the mecC gene is a challenge, since they can be mistakenly classified as sensitive or borderline depending on the technic used for their identification, and can present resistant to cefoxitin while being sensitive to oxacillin (PATERSON; HARRISON; HOLMES, 2014).
Strains that have the mecC gene has similar high dissemination potential to strains with mecA gene and have also been described for isolates obtained from humans, production and wild animals, wastewaters, waters of sewage treatment stations. The mecC gene can also be present in MRCoNS strains (BECKER; HEILMANN;PETERS, 2014;PETERSEN et al., 2013;PORRERO et al., 2014). Pigs have been reported as significant reservoirs of MRSA and MRNAS strains, which have drawn attention of researchers that seek a better understanding of epidemiological aspects involving their transmissibility to humans and animals and development of resistance to antibiotics in these microorganisms (BUTAYE; ARGUDÍN;SMITH, 2016;NORMANNO et al., 2015;SMITH et al., 2013;SOUZA et al., 2012;VERKADE;KLUYTMANS, 2014).
Staphylococcus spp. are present in all pig production processes until the slaughter and the final consumer. Microbiological analysis from nasal mucus, skin, tonsils, feces, and internal organs of apparently healthy pigs at slaughter showed presence of S. aureus, denoting the importance of this animal species as a reservoir of these microorganisms (FRANA et al., 2013;HERMANS et al., 2010;O'SULLIVAN et al., 2011;SCHLEIFER;BELL, 2015;TENHAGEN et al., 2009).
The presence of Staphylococcus spp. in pigs has not been limited to the colonization of these animals. Changes in production systems from open pastures and low animal density to a more dense and intensive system made the animal environment also a reservoir of Staphylococcus spp. The animals in a production system can have contact with the same surfaces; thus, water drinkers, feeders, and the air and dust deposited in their environment become sources of contagion for all animals and handlers. The formation of bioaerosols causes risks of secondary exposition, which can lead to contaminations outside the production site (BARCELLOS et al., 2009;DAVIS et al., 2018;FELD et al., 2018).
The direct contact of handlers with animals that carry Staphylococcus spp. is also an important form of transmission of these microorganisms from pigs to humans, exposing them to the colonization of microorganisms and possible infections. These handlers are in a high-risk class, with frequency of contamination of 15% to 37.8%, which is higher than that for workers of slaughterhouses, who have less contact with the animals and are trained for good hygiene practices while handling food (CUI et al., 2009;MASSON;CARVALHO, 2012;PARISI et al., 2019;SCHMIDT;KOCK;EHLERS, 2015;SMITH et al., 2013).
However, the origin of S. aureus is not associated exclusively with pigs or to their environments of growth and slaughter, or to handlers of these animals, but also to handlers that assist in the production and processing of meat products. Persistent or intermittent nasal colonization by S. aureus have been found in 30% to 50% of healthy individuals (SERGELIDIS; ANGELIDIS, 2017;CASTRO et al., 2016;SEZER et al., 2015;COSTA et al., 2015;FERREIRA et al., 2014;KLUYTMANS, 2010).
The presence of S. aureus in raw or processed pig meat and the contamination of these bacteria between food handlers and these foods have been described in the literature (BUYUKCANGAZ et al., 2013;CASTRO et al., 2016;CHON;SUNG;KHAN, 2017;COSTA et al., 2015;FALL et al., 2012;SEZER et al., 2015).
The importance of investigating S. aureus strains in animal production, including pigs, denotes the hygienicsanitary quality of production systems and the potential of these strains to of staphylococcal enterotoxins. These superantigens are involved with staphylococcal food intoxication, which is one of the diseases transmitted by food that presents high occurrence worldwide. The production of staphylococcal enterotoxins increases the pathogenic potential of these bacteria (DIEDRICH et al., 2013;FERREIRA et al., 2014;HENNEKINNE et al., 2010;KLUYTMANS, 2010;SERGELIDIS;ANGELIDIS, 2017) Therefore, considering the production chain of pig meat and the involved animals, environments, handlers, workers, and equipment and utensils, there are several different sources of contamination and transmission of Staphylococcus spp., which denotes the importance of this animal production for public health (DWEBA; ZISHIRI; EL ZOWALATY, 2019;FERGUSON et al., 2016;MASSON;CARVALHO, 2012;VAN CLEEF et al,. 2010).
Before the recognition of pigs and other production animals as reservoirs of MRSA strains, little interest was perceived for researches about S. aureus in these animals (LINHARES et al., 2015;PANTOSTI, 2012). This is evidenced by the many works that researched only the presence of MRSA strains in these animals (LASSOK;TENHAGEN, 2013;PANTOSTI, 2012;SUN et al., 2015).
Pigs have been described as the most important reservoir and ecosystem for the development of resistance S. aureus strains to antimicrobial agents (BUTAYE; ARGUDÍN; SMITH, 2016). Price et al. (2012) showed that the LA-MRSA ST398 developed from a clone of human MSSA that colonized pigs and acquired the genetic determinant SCCmec. The origin and transmissibility of SCCmec for S. aureus involve the MRCoNS, whose presence in pigs is also widely reported (BECKER; HEILMANN;PETERS, 2014;TULINSKI et al., 2012).
The presence of the clone LA-MRSA ST 398 in Brazil was first reported in cows with mastitis (SILVA et al., 2014). Its occurrence in pigs was first described in a case of exudative epidermitis in the state of Rio Grande do Sul, and the strain already presented an intermediate resistance to the glycopeptide vancomycin (MORENO et al., 2016).
The first description of this microorganism in humans involved a patient with cystic fibrosis, whose infection probably occurred after a visit to a rural propriety, where the patient had recreational contact with animals of that environment (LIMA et al., 2017). However , André Neto et al. (2017) described nasal colonization by strains of the clone MRSA ST398 in six children, which presented no apparent risk factor-did not live in rural environments or had contact with animals. This result denotes the potential of this MRSA, which emerged in pigs, to disseminate and overcome ecological barriers.
Currently, the frequency of MRSA strains that belong to the clone LA-MRSA ST398 in Brazil appears to be low, and no reports of strains containing the mecC gene are found. However, a recent study on the use of antimicrobial agents in 25 pig production systems and on detection of MRSA strains showed the presence of these microorganisms in 80% of the systems evaluated, with 68.0% of pigs hosting LA-MRSA ST398 strains (DUTRA, 2017). Therefore, the evident high capacity of dissemination and genomic plasticity for the development of different mechanisms of resistance to antibiotics make essential the oversight of multiresistant strains of Staphylococcus spp.; and these researches should involve pets and wild and production animals, and the whole food production chain for human consumption, with especial attention to pig farming.

CONCLUSIONS
The emergence and wide dispersion of methicillinresistant Staphylococcus aureus and methicillin-resistant non aureus Staphylococci because of inadequate use of antimicrobial agents in pig farming denote the epidemiological involvement of this animal species with the transmission of these microorganisms and occurrence of infections in humans and animals all around the world.
The risk of dissemination of these microorganisms is particularly high in countries where legislation, regulatory oversight, and monitoring systems for the use of antimicrobial agents and prevention and control of antimicrobial resistance are weak or inadequate. Brazil fits this profile; thus, the public health policies involving actions that generate a careful and responsible use of antibiotics are essential for the preservation of the efficacy of these medicines and to support the One Health approach.